human il 12 p70 duosettm elisa development systems  (R&D Systems)

Journal: bioRxiv

Article Title: BLAST: A blue light-assisted secretion toolkit tunable by reversible protein-protein interactions

doi: 10.64898/2026.03.30.715452

Figure Lengend Snippet: Secretion of therapeutic proteins with a-BLAST and d-BLAST. (a) The human preproinsulin (preproINS) construct contains a signal peptide, B-chain (yellow), C-peptide (dark gray), and A-chain (yellow), with engineered Furin cleavage sites flanking the C-peptide for maturation. To facilitate efficient processing within the Golgi apparatus, Furin protease was co-transfected with the BLAST modules. (b) Kinetic profiling of light-induced insulin secretion. Summary graphs of secreted C-peptide levels, quantified by ELISA, as a proxy for insulin secretion from a-BLAST (left) and d-BLAST (right). Both systems exhibited significant, time-dependent insulin release starting from 2 h of illumination (8.2-fold for a-BLAST, 8.4-fold for d-BLAST), reaching maximal induction at 24 h (13.8-fold for a-BLAST, 19.3-fold for d-BLAST). (c) Plasmid configurations for IL-12 secretion. Schematic of the heterodimeric cytokine IL-12-a-BLAST (left) and d-BLAST-IL-12 (right) constructs. (d) Kinetic profiling of light-induced IL-12 secretion. Summary graphs showing IL-12 secretion levels measured by ELISA. Significant secretion was observed starting at 3 h for a-BLAST (2.5-fold) and 2 h for d-BLAST (2.2-fold). At the 24 h time point, d-BLAST (4.7-fold) demonstrated a slightly higher dynamic range compared to a-BLAST (4.5-fold). Open circles represent individual measurements from three biologically independent samples. Data are presented as means ± S.D. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test (ns = not significant, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Similarly, IL-12 secretion was measured using a Human IL-12 p70 ELISA Kit (R&D Systems; Catalog #D1200) according to the manufacturer’s instructions.

Techniques: Construct, Transfection, Enzyme-linked Immunosorbent Assay, Plasmid Preparation

Journal: bioRxiv

Article Title: BLAST: A blue light-assisted secretion toolkit tunable by reversible protein-protein interactions

doi: 10.64898/2026.03.30.715452

Figure Lengend Snippet: Functional validation of optogenetically secreted IL-12 using a reporter cell assay. (a) Schematic of the experimental workflow for bioactivity validation. The protocol spans 5 days. HEK293T cells transfected with BLAST-IL-12 were subjected to dark or blue light conditions for 24 h (Day 3). On Day 4, the conditioned media containing secreted IL-12 was harvested and transferred to HEK-Blue™ IL-12 reporter cells (seeded on Day 3). After a 24 h incubation to allow for signal transduction, SEAP activity was quantified (Day 5). (b) Plasmid configurations. Schematics of the a-BLAST-IL-12 (upper) and d-BLAST-IL-12 (lower) constructs used for the assay. (c) Illustration of the JAK-STAT signaling pathway in the reporter cells. Binding of secreted IL-12 to the IL-12 receptor complex activates Tyk2/JAK2, leading to STAT4 phosphorylation. Phosphorylated STAT4 dimerizes and translocates to the nucleus to induce SEAP expression. (d) Summary graph of SEAP activity induced by the conditioned media. The results confirm that both systems secrete biologically active IL-12 upon blue light stimulation, exhibiting robust fold changes (12.4-fold for a-BLAST and 11.3-fold for d-BLAST) compared to the dark control. Data are presented as means ± S.D. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test (**** P < 0.0001).

Article Snippet: Similarly, IL-12 secretion was measured using a Human IL-12 p70 ELISA Kit (R&D Systems; Catalog #D1200) according to the manufacturer’s instructions.

Techniques: Functional Assay, Biomarker Discovery, Transfection, Incubation, Transduction, Activity Assay, Plasmid Preparation, Construct, Binding Assay, Phospho-proteomics, Expressing, Control